a) Field of the Invention
The present invention relates to an optical apparatus for irradiating a sample with light and detecting light, fluorescent light in particular, emitted from the sample. Furthermore, the present invention relates to an optical apparatus which is capable of detecting fluorescent light having a plurality of wavelengths at the same time.
b) Description of the Prior Art
For biological researches in the recent years, functions of living bodies have been studied using not fixed samples but living samples. Speaking concretely of the biological researches, functions of protein molecules are clarified by attaching the protein molecules which attract attentions to fluorescent molecules which attach specifically to the protein molecules, and observing and analyzing movements and distributions of the molecules using fluorescence microscopes. In addition, fluorescent substances which have recently appeared make it possible to allow light emitting proteins to exist in cells, thereby observing and analyzing proteins and the like in conditions where physiological activities are kept better. Furthermore, attempts are being made to measure movements and distributions of a plurality of molecules at the same time using a plurality of fluorescent dyes for clarifying higher functions in cells.
Described in Cell Engineering (Vol. 17, No. 6, pp956 to 965) is an apparatus which is configured to irradiate cells with excitation light at intensities as low as possible, thereby lessening damages on cells in order to make it possible to observe the cells in living conditions.
The apparatus described in this literature uses a light attenuating filter (ND filter) which is disposed in an illuminating optical system for weakening the excitation light.
Importance of the ND filter in observation of such fluorescent substances and a density of the ND filter appropriate for observing living cells are described in xe2x80x9cGFP and Bio Imagingxe2x80x9d (separate volume of Experimental Medicine, Experiment Course in Post Genome Age 3, 2000, p156 issued by Youdo, Japan).
Furthermore, observing apparatuses which are used for observations with a plurality of fluorescent dyes are disclosed by Japanese Patents Kokai Publication No. Hei 8-320437, No. Hei 11-52252 and No. Hei 11-223773.
The apparatus disclosed by Japanese Patent Kokai Publication No. Hei 8-320437 uses a dichroic mirror for multiple excitation such as triple excitation or quadruple excitation as a dichroic mirror for observing a plurality of fluorescent light at the same time. There is available a dichroic mirror which has a spectral characteristic shown in FIG. 19. This dichroic mirror is manufactured by a method which deposits a thin film which is referred to as the so-called soft coat film while maintaining a substrate in the vicinity of normal temperature in order to obtain the dichroic mirror having the spectral characteristic shown in FIG. 19. Since this soft coat film has low resistance to temperature and humidity variations, chemicals and physical impacts such as that caused by wiping at a surface cleaning time, the spectral characteristic of the soft coat film is often deteriorated with time lapse and the soft coat film requires a remarkably high cost for maintenance.
Furthermore, a technique for observing samples multiply dyed with fluorescent dyes is described in a published bulletin of Japanese Patent Kokai Publication No. Hei 11-52252. However, the technique described in this bulletin is configured to allow independent dichroic mirrors suited to the fluorescent dyes to be used in exchange without using a dichroic mirror for multiple excitation. Accordingly, this technique does not permit observing movements and distributions of a plurality of molecules using a plurality of fluorescent dyes at the same time. Furthermore, an observing apparatus for observation by this technique allows vibrations to be produced when the dichroic mirrors are exchanged at a high speed since the apparatus uses a dichroic mirror holding member which is more complicated and larger than an excitation filter and as absorption filter. Furthermore, the apparatus requires preparing the dichroic mirrors in a number of the fluorescent dyes for observing the plurality of fluorescent dyes at the same time, thereby posing a problem of cost. Furthermore, differences among parallelisms of the dichroic mirrors produce deviations among observed fluorescent images. Means for canceling the deviations which is described in this bulletin is extremely complicated and it is difficult to cancel the deviations by simple and inexpensive means.
Furthermore, Japanese Patent Kokai Publication No. Hei 11-223773 discloses a technique which is configured to permit observing a plurality of fluorescent images at the same time using a plurality of dichroic mirrors disposed in directions of optical axes of an objective lens and an observing optical system, but this technique requires a plurality of light sources.
The present invention has been achieved in circumstances described above and has an object to provide as optical apparatus which is configured to permit simultaneously observing a plurality of fluorescent dyes used for clarifying functions of living bodies.
In order to accomplish the above described object, an optical apparatus according to the present invention comprises a light source, an illuminating optical system for illuminating a sample with illuminating light from the light source, a first wavelength selecting member disposed after the illuminating optical system, a light beam splitter for directing the illuminating light from the light source to the sample, an objective lens disposed between the light beam splitter and the sample, a detector for detecting light from the sample through the objective lens and a second wavelength selecting member disposed in the objective lens system or between the objective lens and the detector, the first wavelength selecting member has at least a wavelength selecting element which selectively transmits light having wavelengths in a predetermined region, the second wavelength selecting member has at least another second wavelength selecting element which transmits light having a predetermined wavelength out of light reflected by the sample or emitted from the sample, and the light beam splitter has an optical element which has a transmitting-reflecting characteristic of transmittance not lower than 85% and reflectance not higher than 15% at least in a wavelength region from 400 nm to 700 nm.
An optical apparatus according to the present invention has a configuration like that described above, in which an optical element of a light beam splitter has a shape of a plane parallel plate and an antireflection coating at least on a surface (second surface) located on a side opposite to a surface (first surface) on which illuminating light are incident from a light source.
In the optical apparatus according to the present invention, the optical element of the light beam splitter may have the antireflection coatings on both the first surface and the second surface.
Furthermore, an optical apparatus according to the present invention is configured to have either of the configurations described above, use a light beam splitter which further comprises a dichroic mirror and allow the optical element to be switched to the dichroic mirror.